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Telotaggg telomerase pcr elisa plus kit

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The TeloTAGGG Telomerase PCR ELISA PLUS kit is a laboratory product designed for the quantitative determination of telomerase activity in cell extracts. It utilizes a highly sensitive and specific method to measure telomerase activity.

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37 protocols using telotaggg telomerase pcr elisa plus kit

1

Telomerase Activity Measurement by TRAP

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Telomerase activity was measured by a telomeric repeat amplification protocol (TRAP) assay with the TeloTAGGG Telomerase PCR ELISA plus Kit (Roche Molecular Biochemicals, Germany). According to the manufacturer’s protocol, approximately 1 × 106 cells were harvested for each reaction and centrifuged at 12,000 ×g for 20 min at 4 °C. TRAP reaction products were generated in the supernatant after amplification and incubated with the sealant at 37 °C for 1 h. Anti-DNP antibody was added and incubated for 30 min in dark. After adding TMB solution and termination solution, the absorbance for the final product was acquired by measuring absorbance at 450 and 690 nm. The difference value (OD = A450-A690) stands for telomerase activity.
Lin L., Zhang L., Li X.T., Ji J.K., Chen X.Q., Li Y.L, & Li C. (2020). Rhynchophylline Attenuates Senescence of Endothelial Progenitor Cells by Enhancing Autophagy. Frontiers in Pharmacology, 10, 1617.
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2

Telomerase Activity Assay Protocol

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Telomerase activity was determined using the TeloTAGGG telomerase PCR ELISA Plus kit (Roche, Basel, Switzerland). Cells (2 × 105) treated with Costunolide in the presence or absence of NAc or animal tissues were lysed according to the manufacturer's instruction. Equal amounts of protein (0.5 μg) from cell and tissue extract were used. The assay was performed according to the manufacturer's protocol as described previously.27 (link)
Ahmad F., Dixit D., Sharma V., Kumar A., Joshi S.D., Sarkar C, & Sen E. (2016). Nrf2-driven TERT regulates pentose phosphate pathway in glioblastoma. Cell Death & Disease, 7(5), e2213-.
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3

Telomerase Activity Quantification in GBM

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The enzymatic activity of telomerase was measured using TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche) according to the manufacturer’s protocol. GBM tissues and cells were homogenized in ice-cold lysis buffer using automill (Tokken). Briefly, after BCA protein quantification of the lysates, 10 µg of proteins were incubated in total volume of 50 µl reaction mixture at 25 °C for 30 min to allow the telomerase to add telomeric repeats to the end of the biotin-labeled primer. Consequently, PCR was conducted for 33 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s, followed by additional extension time of 10 min at 72 °C and holding at 4 °C. The telomerase activity was measured at 450 nm and the reference wavelength 690 nm. Relative telomerase activity (RTA) of each sample was calculated according to the instruction of TeloTAGGG Telomerase PCR-ELISA PLUS Kit.
Kim S., Seo Y., Chowdhury T., Yu H.J., Lee C.E., Kim K.M., Kang H., Kim H.J., Park S.J., Kim K, & Park C.K. (2020). Inhibition of MUC1 exerts cell-cycle arrest and telomerase suppression in glioblastoma cells. Scientific Reports, 10, 18238.
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4

Telomerase Activity in T-Cell Subtypes

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For determination of telomerase activity naïve and memory CD4+ and CD8+ T-cells were isolated and cultured in advanced RPMI 1640 (GIBCO) in the presence of 30 U/mL rIL-2 (Sigma, Vienna, Austria) and CD3/CD28 Dynabeads (bead:cell ratio 1:1; Invitrogen). After 3 and 6 days, 2 × 105 cells were harvested and telomerase activity was determined using TeloTAGGG Telomerase PCR ELISAplus kit (Roche) according to ‘Telomeric Repeat Amplification Protocols (TRAP)’.21 (link)
Fessler J., Raicht A., Husic R., Ficjan A., Duftner C., Schwinger W., Dejaco C, & Schirmer M. (2015). Premature senescence of T-cell subsets in axial spondyloarthritis. Annals of the Rheumatic Diseases, 75(4), 748-754.
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5

Quantifying Cellular Markers in Mouse Tissues

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Western blotting was used to detect and quantify TERT, p53, and p21 in mouse tissues and PA-SMCs. The antibodies used were anti-TERT (Santa-Cruz Biotechnology, Nanterre, France), anti-p53 (Cell Signaling Technology, Boston, MA), and monoclonal anti-p21Waf1/Cip1 (Cell Signaling Technology) antibodies. Levels of TERT, p21, and Bax mRNAs in lung tissue were determined by use of a real-time quantitative polymerase chain reaction assay (PCR). Total mRNA was extracted from tissues with the RNeasy Mini Kit (Qiagen, ZA Courtaboeuf, France). Real-time quantitative PCR was performed in a 7900HT real-time PCR system (Applied Biosystems, ZA Courtaboeuf, France), using SYBR Green Mix (Invitrogen) as described previously.20 (link) The amounts of mRNA were normalized for the amount of the control gene, 18s, which was chosen among 4 tested housekeeping genes on the basis of its stable expression in all samples (data not shown). Because the control gene might amplify genomic DNA, contaminating the RNA samples, we checked the absence of such contamination by performing the reverse-transcriptase step with and without reverse transcriptase.
The endogenous enzymatic activity of telomerase in mouse lung and cultured cells was quantified with the teloTAGGG telomerase PCR Elisaplus kit (Roche Diagnostics, Meylan, France).
Mouraret N., Houssaïni A., Abid S., Quarck R., Marcos E., Parpaleix A., Gary-Bobo G., Dubois-Randé J.L., Derumeaux G., Boczkowski J., Delcroix M., Blasco M.A., Lipskaia L., Amsellem V, & Adnot S. (2014). Role for Telomerase in Pulmonary Hypertension. Circulation, 131(8), 742-755.
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6

Telomerase Activity Quantification in POMECs

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Telomerase activity of primary POMECs and hTERT-POMECs was analyzed using the Telo TAGGG Telomerase PCR ELISAPLUS kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Telomerase-positive HeLa cells were used as a positive control and a negative control with enzyme-inactivated sample material was performed according to the instructions. Briefly, the extract of equivalent cells was harvested for polymerase chain reaction (PCR) amplification. Then the PCR products were denatured and hybridized to digoxigenin-labeled detection probes for telomeric repeat-specific ELISA. The absorbance of samples was measured with a microplate spectrophotometer (Infinite 200 PRO Nano-Quant, Tecan, Switzerland) at 450 and 690 nm.
Cui H., Liang W., Wang D., Guo K, & Zhang Y. (2019). Establishment and Characterization of an Immortalized Porcine Oral Mucosal Epithelial Cell Line as a Cytopathogenic Model for Porcine Circovirus 2 Infection. Frontiers in Cellular and Infection Microbiology, 9, 171.
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7

Quantifying Telomerase Activity in SH-SY5Y Cells

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We measured the telomerase activity followed by the method described before (Mookherjee et al., 2007 (link)). In brief, the 3‐((3‐cholamidopropyl) dimethylammonio)‐1‐propanesulfonate buffer was used to lyse SH‐SY5Y cells for 30 min on the ice. Then the lysate was centrifuged at 15,000 g for 90 min. Isolated protein was quantified using the bicinchoninic acid (BCA) method, and the TeloTAGGG Telomerase PCR ELISA Plus Kit (Roche, Switzerland) was then utilized to detect the activity of telomerase. Subsequently, the telomeric repeat amplification protocol–PCR reaction system was introduced together with samples and primers. Lastly, RT‐PCR was used to perform the quantification.
Wang P., Wang P., Luan H., Wu Y, & Chen Y. (2022). Midazolam alleviates cellular senescence in SH‐SY5Y neuronal cells in Alzheimer's disease. Brain and Behavior, 13(1), e2822.
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8

Telomerase Activity Quantification by ELISA

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The telomerase activity was analyzed using a TeloTAGGG Telomerase PCR ELISA Plus Kit (Roche, Mannheim, Germany), according to the manufacturer's instructions. Using the ELISA method, the amplified products were immobilized on streptavidin-coated microtiter plates via biotin-streptavidin interaction. Thereafter the amplifications were detected by anti-digoxigenin antibodies conjugated to peroxidase. After addition of the peroxidase substrate (3, 3', 5, 5'-tetramethylbenzidine), the amount of TRAP products was determined by measurement of absorbance at 450 nm using a microplate reader.
Park J.B., Byun C.H, & Park E.Y. (2015). Rat Notochordal Cells Undergo Premature Stress-Induced Senescence by High Glucose. Asian Spine Journal, 9(4), 495-502.
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9

Telomerase Activity Quantification by TRAP Assay

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Telomerase activity was determined by TRAP assay using the TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's protocol. Treated cells were collected and lysed in ice-cold lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min. Following centrifugation (10,000 × g for 10 min at 4°C), the supernatants were collected and stored at −80°C, and ~5 µl of each supernatant was added to the PCR reaction mixture provided in the kit. PCR was then carried out under the following reaction conditions: Primer elongation (25°C for 30 min), telomerase inactivation (94°C for 5 min), 30 cycles of amplification (94°C for 30 sec, 50°C for 30 sec, 72°C for 90 sec) and extension (72°C for 5 min). The PCR product was denatured and hybridized with the digoxigenin (DIG)-labeled telomeric repeat-specific probe. Finally, the PCR product was detected using the anti-DIG-POD antibody (1:5,000, incubated at 37°C for 60 min) and measured at 450 nm on a microplate reader.
Huang L., Jin K, & Lan H. (2019). Luteolin inhibits cell cycle progression and induces apoptosis of breast cancer cells through downregulation of human telomerase reverse transcriptase. Oncology Letters, 17(4), 3842-3850.
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10

Quantifying Telomerase Activity in Hepatocellular Carcinoma

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Hepatocellular carcinoma cells (1 × 105) and tumor‐forming tissue from mice were lysed and centrifuged at 16 000 g for 20 minutes at 4°C. The supernatant was gently collected and the protein concentration in the supernatant was determined by a BCA protein assay kit (Pierce Biotechnology). A quantitative TeloTAGGG Telomerase PCR ELISA plus kit (Roche Applied Science) was used to evaluate telomerase activity according to the manufacturer’s recommendation. Results were obtained with at least 3 replicates.
Cao H., Zhai Y., Ji X., Wang Y., Zhao J., Xing J., An J, & Ren T. (2020). Noncoding telomeric repeat‐containing RNA inhibits the progression of hepatocellular carcinoma by regulating telomerase‐mediated telomere length. Cancer Science, 111(8), 2789-2802.
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